PROJECT SUMMARY Glucagon-like peptide-1 (GLP-1) enhances islet function by potentiating glucose-stimulated insulin secretion (GSIS) from pancreatic ?-cells; however, the mechanisms by which GLP-1 potentiates GSIS remain incompletely defined. In the classic model, GLP-1 secreted by the intestinal L cells in response to the ingestion of nutrients stimulates the ?-cell GLP-1 receptor (GLP-1R) to enhance GSIS. This model is currently in question, as the short half-life of GLP-1 and its rapid degradation present limitations as to how GLP-1 can mediate effects in distant targets, such as pancreatic ?-cells. In explanation of such limitations, there is an emerging hypothesis suggesting that GLP-1 locally produced by ?-cells acts in a paracrine manner on neighboring ?-cells to stimulate GSIS, which ultimately promotes lowering of glycemia. Proglucagon is expressed in the gut and ?-cells and is cleaved to form GLP-1 or the counter-regulatory hormone, glucagon, depending on the prohormone convertase (PC) type present. It was previously thought that the tissue-specific processing of proglucagon was due to the differential expression of PC1/3 and PC2, but several studies in rodent models and humans have shown that ?- cells can produce active GLP-1 and express PC1/3. However, the mechanisms by which ?-cell PC1/3 expression is regulated are unknown. Identifying a mechanism to increase ?-cell PC1/3 expression to increase GLP-1 production at the expense of glucagon will provide a powerful therapeutic modality for the regulation of blood glucose concentrations in patients with T2DM. We have shown that increased ?-cell GLP-1R signaling increases ?-cell PC1/3 and GLP-1 expression. My preliminary data suggest that insulin may serve as potential intermediate by which ?-cell GLP-1R signaling alters ?-cell proglucagon processing. Specifically, I hypothesize that ?-cell GLP-1R signaling increases ?-cell PC1/3 expression through both ?-cell insulin receptor (IR) dependent and independent pathways. In Aim 1, I will assess the role of ?-cell IR signaling to determine whether insulin is necessary to alter the secretory phenotype of ?-cells by measuring glucose regulation, GSIS, and PC1/3 expression in ?-cell-specific Ir?-cell +/+ and Ir?-cell -/- mice with and without stimulation by exogenous GLP-1. In Aim 2, I will determine the pathways through which ?-cell GLP-1R signaling increases ?-cell PC1/3 expression by single-cell RNA-sequencing of islets from inducible ?-cell-specific Glp-1r?-cell+/+ and Glp-1r-cell-/- mice, as well as human islets with ?-cell-specific GLP-1R knockout. Together, these studies will define a link between the pathways regulating ?-cell PC1/3 expression and GLP-1 production; thus, enabling targeting of the ?-cell secretory phenotype for the treatment of T2DM.